I. Membrane lipids are not just a filler or an inert solvent for membrane proteins; they are functionally involved. Interactions between lipids and embedded proteins control conformational equilibrium between different functional states of proteins. In order to examine the influence of lipid packing energetics on ion channel expression, we have studied the relative probabilities of alamethicin channel formation in dioleoylphosphatidyl serine (DOPS) bilayers as a function of pH. The rationale for this strategy is our earlier finding that the higher-conductance states, corresponding to larger polypeptide aggregates, are more likely to occur in the presence of lipids prone to hexagonal phase formation (specifically DOPE), than in the presence of lamellar lipids (DOPC). X-ray diffraction studies on DOPS show a progressive decrease in the intrinsic curvature of the constituent monolayers as well as a decreased probability of hexagonal phase formation when the charged lipid fraction is increased. We have explored how proton titration of DOPS affects lipid packing energetics, and how these energetics couple titration to channel formation. In low ionic strength NaCl solutions at neutral pH, the open channel in DOPS membranes spends most of its time in states of lower conductance and resembles alamethicin channels in DOPC; at lower pH, where the lipid polar groups are neutralized, the channel probability distribution resembles that in DOPE. This correlation, made for the charged lipid DOPS, agrees with the logic of our conclusions drawn from earlier observations on alamethicin in mixtures of neutral lipids. There is a pronounced, 50-fold effect on channel state to state transitions from a pH shift of about two units. This finding probably unveils an additional, previously unrecognized mechanism of pH regulation in membrane transport. II. The geometry of an ion channel pore can be probed with differently sized water-soluble polymers. If they are small enough, polymers can go inside the ion channel pore. This is seen as a decrease in channel conductance upon polymer addition to the membrane-bathing solution. Asymmetric (one-sided) application of penetrating polymers, polyethylene-glycols (PEGs), to a well-defined channel formed by Staphylococcus aureus alpha-toxin has been shown to gauge channel pore geometry in more detail than their symmetrical (two-sided) application. Polymers added to the cis- side of the planar lipid membrane (the side of protein addition) affect channel conductance differently than polymers added to the trans- side. We estimate the radii of the two openings of the channel as practically identical and equal to 1.2-1.3 nm. Two apparent constrictions with radii ~0.9 nm and ~0.6-0.7 nm are inferred to be present in the channel lumen, the larger one being closer to the cis- side. These structural findings agree well with crystallographic data on the channel structure and verify the practicality of polymer probing. The general features of PEG partitioning have been examined using available theoretical considerations assuming there is no attraction between PEG and the channel lumen. It is shown that the sharp dependence of partition coefficient on polymer molecular weight found under both symmetric and asymmetric polymer application can be rationalized within a hard sphere non-ideal solution model. This finding is rather surprising since PEG forms highly flexible coils in water with a Kuhn length of only several Angstroms. - Ion channels, reconstitution, regulation, metabolite selectivity, signal transduction, stochastic resonance.